Southern tick-associated rash illness (STARI) is a newly described Lyme disease-like infection reported from the southeastern and south central US caused by Borrelia lonestari. Our long-range goal is to understand the importance of B. lonestari as a human pathogen. The objective of this application is to develop direct microscopic, culture, molecular, and serologic methods that will allow diagnosis of B. lonestari infection in humans, experimental animal models, and reservoir hosts. Borrelia lonestari is most closely related to B. miyamotoi, B. theileri, and the relapsing fever Borrelia, all of which produce high-density spirochetemia during infection. Infection with relapsing fever Borrelia also results in a strong humoral immune response. Our pilot trial infection of two deer fawns showed that B. lonestari also produces a circulating spirochetemia and results in seroconversion of infected animals. The rationale behind the proposed research, therefore, is that like infection with relapsing fever Borrelia spp., B. lonestari infection is likely to result in detectable spirochetemia, allowing culture of the organism from blood, visualization on blood smears, and detection by molecular assays, as well as a strong antibody response which will permit development of immunodiagnostic tools. To accomplish the objectives of this application, we will pursue three specific aims: (1) refine our methods for culturing B. lonestari and for directly identifying spirochetes in circulating blood using samples collected from experimentally infected deer; (2) develop molecular assays for identification of Borrelia lonestari that allow it to be quickly and reliably distinguished from B. burgdorferi and relapsing fever Borrelia; and (3) identify candidate antigens that may elicit production of B. lonestari-specffic antibodies. At the completion of this research, we expect to have developed culture, microscopic, molecular, and serologic techniques that will specifically identify B. lonestari infection. The diagnostic assays resulting from this proposed research will allow accurate identification of the Borrelia spp. causing disease in people and, ultimately, a more robust understanding of the role of B. lonestari as a human pathogen. Improved diagnosis of human B. lonestari infection should result. [unreadable] [unreadable] [unreadable]